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Dominant-Negative and Targeted Null Mutations in the Endothehal Receptor Tyrosine Kinase, Tek, Reveal a Critical Role in Vasculogenesis of the Embryo

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Dominant-Negative and Targeted Null Mutations in the Endothehal Receptor Tyrosine Kinase, Tek, Reveal a Critical Role in Vasculogenesis of the Embryo Downloaded from genesdev.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Dominant-negative and targeted null mutations in the endothehal receptor tyrosine kinase, tek, reveal a critical role in vasculogenesis of the embryo Daniel J. Dumont, 1's'6 G4rard Gradwohl, 2 Guo-Hua Fong, 1 Mira C. Puri, 1'3 Marina Gertsenstein, 1 Anna Auerbach, 1 and Martin L. Breitman 1'3'4 ~Division of Molecular and Developmental Biology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada; 2Laboratoire de G6n&ique Mol&ulaire des Eucaryotes, Centre National de la Recherche Scientifique (CNRS), Unit6 184 de Biologie Mol6culaire et de G4n6tique, Institut National de la Sant6 et de la Recherche M4dicale (INSERM), Institut de Chimie Biologique, Facult6 de M6decine, Strasbourg, France; 3Department of Molecular and Medical Genetics, University Toronto,Toronto, Ontario, Canada The receptor tyrosine kinases (RTKs) expressed on the surface of endothelial cells are likely to play key roles in initiating the program of endothelial cell growth during development and subsequent vascularization during wound healing and tumorigenesis. Expression of the Tek RTK during mouse development is restricted primarily to endothelial cells and their progenitors, the angioblasts, suggesting that Tek is a key participant in vasculogenesis. To investigate the role that Tek plays within the endothelial cell lineage, we have disrupted the Tek signaling pathway using two different genetic approaches. First, we constructed transgenic mice expressing a dominant-negative form of the Tek receptor. Second, we created a null allele of the tek gene by homologous recombination in embryonic stem (ES) cells. Transgenic mice expressing dominant-negative alleles of Tek or homozygous for a null allele of the tek locus both died in utero with similar defects in the integrity of their endothelium. By crossing transgenic mice that express the lacZ reporter gene under the transcriptional control of the endothelial cell-specific tek promoter, we found that the extraembryonic and embryonic vasculature was patterned correctly. However, homozygous tek embryos had -30% and 75% fewer endothelial cells at day 8.5 and 9.0, respectively. Homozygous null embryos also displayed abnormalities in heart development, consistent with the conclusion that Tek is necessary for endocardial/myocardial interactions during development. On the basis of the analysis of mice carrying either dominant-negative or null mutations of the tek gene, these observations demonstrate that the Tek signaling pathway plays a critical role in the differentiation, proliferation, and survival of endothelial cells in the mouse embryo. [Key Words: Receptor tyrosine kinase; endothelial cell growth; Tek; vasculogenesis; transgenic mice; homozygous embryos] Received June 1, 1994; revised version accepted July 7, 1994. The receptor tyrosine kinase {RTK) family of cell-sudace mouse all depend on intact signaling pathways that are proteins is known to play key roles in cell-cell commu- controlled by different members of the RTK family nication in multicellular metazoan organisms (Pawson (Reith and Bernstein 1991; Pawson 1993}. and Bemstein 1991). Genetic and biochemical studies on Five RTKs have been identified whose expression is this large family of proteins have shown that different restricted primarily to cells of the endothelial lineage. RTKs are responsible for transducing important develop- On the basis of similarities in their primary amino acid mental, proliferative, cell survival, and migratory signals sequences, they have been placed into two distinct sub- from the outside to the inside of the cell {Hunter and families of RTKs (Dumont et al. 1993; Hunter and Lind- Lindberg 1994). The development and proper functioning berg 1994). The members of one subfamily, including of cell systems as diverse as the compound eye in the fly, Flt-4 (Pajusola et al. 1992) and the Vascular endothelial the vulva in the nematode, and hematopoiesis in the growth factor (VEGF) receptors Flt-1 (Matsushime et al. 1987; Shibuya et al. 1990) and Flk-1/KDR {Terman et al. 1991; Millauer et al. 1993), are characterized by extra- 4This manuscript is dedicated to the memory of Dr. Martin L. Breitman, cellular domains consisting of seven immunoglobulin- who died on February 13, 1994. SCorresponding author. like loops and an intracellular split kinase domain. The 6present address: AMGEN Institute, Toronto Ontario MSG 2C1, Canada. second subfamily contains the two receptors Tek and GENES & DEVELOPMENT 8:1897-1909 91994 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/94 $5.00 1897 Downloaded from genesdev.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Dumont et al. Tie (Wilks 1989; Dumont et al. 1992, 1993; Partanen et pathway by expressing a dominant-negative form of Tek al. 1992; Iwama et al. 1993; Maisonpierre et al. 1993; in transgenic mice. Previous studies have shown that Sato et al. 1993; Ziegler et al. 1993), both of whom have structural mutations that abolish or diminish the intrin- complex extracellular domains consisting of two immu- sic tyrosine kinase activity of other RTKs act in a dom- noglobulin-like loops separated by three EGF-like re- inant-negative manner in both cells and in the intact peats that are followed by three fibronectin type III-like organism. For example, naturally occurring mutations in repeats. The intracellular portions of Tek and Tie also the Kit RTK in the dominant white-spotting (W) mouse contain a split kinase domain. mutant act in a dominant-negative manner to inhibit The differing spatial and temporal patterns of expres- melanogenesis, hematopoiesis, and gametogenesis sion of these RTKs during development suggest that they (Reith et al. 1990). Similarly, expression of other RTKs play different roles within the endothelial cell lineage. with mutations that reduce their intrinsic tyrosine ac- Very early in development [embryonic day 7.0 (E7.0)], tivity act in a dominant-negative fashion to inhibit the ilk-1 is expressed in both the extraembryonic and em- signaling through the corresponding wild-type receptor bryonic mesoderm in regions thought to give rise even- in mammalian cell lines (Reith et al. 1993), in frog em- tually to the embryonic vasculature (Millauer et al. bryos (Amaya et al. 1991), in transgenic mice (Ray et al. 1993; Yamaguchi et al. 1993; D. Dumont, G.-H. Fong, 1991), and in tumor angiogenesis (Millauer et al. 1994). M.C. Puri, G. Gradwohl, K. Alitalo, and M. Breitman, in Second, we created a tek null allele by homologous re- prep.), tek can then be detected in regions that overlap combination in ES cells and introduced the mutation with the flk-l-positive extraembryonic mesoderm at into the mouse germ line. Mice heterozygous for the E7.5, and finally, tie is detected at E8.0 in the flkol- and mutant allele were phenotypically normal, whereas ho- tek-expressing extraembryonic mesoderm (D. Dumont, mozygous mice died in utero at -E9.5 as a consequence G.-H. Fong, M.C. Puri, G. Gradwohl, K. Alitalo, and M. of an underdeveloped vasculature. Embryos for the null Breitman, in prep.). The early onset of ilk-1 expression mutation or hemizygous for the dominant-negative (E7.0) suggests a role for this receptor early in the deter- transgene displayed strikingly similar developmental de- mination of the endothelial cell lineage (Yamaguchi et fects. Taken together, these results demonstrate that the al. 1993), whereas the later, sequential onset of both tek Tek signaling pathway plays a critical role in the vascu- and tie expression suggests that these RTKs may play larization of the mouse embryo. distinct roles during subsequent vascularization of the embryo. Little is known about the expression of fit-1 and fit-4 Results in the mouse. One report has stated that fit-1 is ex- Generation of transgenic mice carrying a tek cDNA pressed in the endothelium of E9.5 embryos (Peters et al. encoding a dominant-negative Tek RTK 1993), but as the onset of expression was not determined in these studies, it is not known whether fit-1 may also To assess rapidly the role of the Tek signaling pathway play an earlier role during vascularization. Although the in mouse development, we introduced a mutation localization and timing of fit-4 expression in the mouse within the tek cDNA that altered the codon for lysine- has not been reported, studies on human fetal tissue 853 to encode an alanine residue. This lysine residue and have reported FLT-4 expression in the epithelium lining its surrounding amino acids are found in a region within the bronchioles of the lung and in the vascular endothe~ the intracellular cytoplasmic domain that is highly con- lium (Pajusola et al. 1992; Kaipainen et al. 1994). Quek- served in all tyrosine kinases, and alteration of this res- 1, the quail homolog of ilk-l, is expressed, as in the idue is known to abolish catalytic function (Nocka et al. mouse, in the mesoderm from the onset of gastrulation, 1990; Reith et al. 1990, 1993). Furthermore, other RTKs whereas the expression of Quek-2, a distinct RTK related that are competent to bind ligand, but lack intrinsic ki- most closely to fit-4, is found later in endothelial cells nase activity, act in a dominant-negative fashion to in- that stain with the endothelial cell-specific antibody hibit signal transduction by wild-type receptor coex- QH1 (Eichmann et al. 1993). pressed in the same cell (Amaya et al. 1991; Ray et al. Our studies have focused on the biological function of 1991; Reith et al. 1993; Millauer et al. 1994). To deter- the routine RTK, Tek, first identified from a cDNA li- mine whether the lysine-to-alanine mutation at codon brary prepared from E12.5 embryonic heart (Dumont et 853 affected the intrinsic tyrosine kinase activity of Tek, al. 1992, 1993). The tek gene encodes a 140-kD protein we introduced this mutated tek cDNA (tek A8s3) into with intrinsic tyrosine kinase activity (Dumont et al.
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